Heart disease is the leading cause of death in the United States. Each year, more than 1 million people suffer from heart attacks. Many of the therapies available to doctors today are aimed at mitigating the damage during heart attacks (e.g., by placing drug-eluting stents in the artery), preventing worsening of symptoms (e.g., by aspirin, beta-blockers, ACE inhibitors, and lipid lowering drugs), or improving blood flow to the heart (e.g., by bypass surgery). These approaches are often ineffective in the long run because heart damage is irreversible, given that the heart generally cannot regenerate new heart muscles. This explains why, despite the best treatments available, patients with severe chronic heart failure have an astounding mortality rate of 20-30% per year. Furthermore, more than 400,000 new cases of congestive heart failure are diagnosed each year. Therefore, there is a great need to find new therapies aimed at the underlying process that will regenerate normal heart tissues in patients.One of the most promising areas of therapeutic research today involves the use of human embryonic stem cells (hESCs). hESCs have the unique ability to self regenerate and to transform into more specialized cell types. For these reasons, researchers are examining whether hESC derived cardiac cells can help regenerate heart cells and improve heart function. Most of the initial studies done in small animals so far have shown that indeed this is the case. However, current methods of differentiating hESC into cardiac cells is still not very efficient. Researchers typically end up with 5-40% of cardiac cells at best. Improving the efficiency and scaling up the production of these hESC-derived cardiac cells is one of our major goals in this grant. Another problem facing clinicians and researchers alike is that they have limited means of monitoring where the hESC-derived cardiac cells go, how long they survive in the heart, when they proliferate, and whether they can transform into heart muscles. Instead, they rely on indirect and often subjective measures for determining efficacy, such as changes in quality of life reported by patients, how long they can exercise on the treadmill, and whether there is any increase in blood flow after stem cell transplant. As for researchers, they have to sacrifice the animals at serial time points to analyze data. This limitation, unfortunately, prevents them from understanding how stem cells work to improve heart function over time. Therefore, we have teamed up with other scientists who use sophisticated imaging devices to track the fate of transplanted cells and process them afterwards using genomics analysis. These efforts will ultimately help explain the mechanism of how hESC-derived cardiac cells can improve cardiac function.
Statement of Benefit to California:
Coronary artery disease is the number one killer in the US. Even under optimal medical management, heart disease patients still frequently suffer adverse consequences such as repeated hospitalizations for chest pain and refractory heart failure symptoms. Stem cell based therapy may one day treat this devastating disease effectively. Human embryonic stem cells (hESC), for example, can transform into cardiac cells and provide an unlimited source of cell supply for cardiac regenerative therapy application. Rather than limited mitigation of symptoms, if hESC fulfills its full potential, it is foreseeable that diseased hearts may be repaired and restored to health. A major challenge to future clinical application of hESC-derived cardiac cells is to scale up production of these cells. Another concern is that these cells may become rejected or may cause tumor after transplantation. Thus, it is crucial to understand how these cells behave inside the living subject before advancing to clinical trials. Our grant is designed to address all 3 questions in a logical and effective manner: (1) how to scale up production of hESC-derived cardiac cells, (2) how to minimize immunogenicity and tumorigencity complications, and (3) how to determine their functional roles in living subjects using novel imaging techniques. Answers to these questions will lead to markedly improved cell transplant protocols that are more reproducible, quantifiable, and beneficial. With our experienced multidisciplinary team members, we are confident that we have the scientific and medical expertise to complete this project.
SYNOPSIS: This is a very broad based proposal to improve on cardiomyocyte differentiation from hESCs, to scale up their production, to created stable lines that express reporters that allow tracking of transplanted cells, to assess tumorigenicity and immunogenicity in transplanted cells, and (!) to examine cardiac function after transplantation of hESC-derived cardiomyocytes. SIGNIFICANCE AND INNOVATION: The area of interest, use of stem cell therapies for coronary artery disease, is probably the most active area of clinical stem cell transplant trials currently, although the bulk of clinical work is really directed at protection of ischemic myocardium rather than regeneration of myocardium. Nonetheless, because the area is so very active, the impact of this particular work may be harder to measure than in less active areas. The problem is certainly important, and the group as a whole has put together the expertise to tackle the important barriers to clinical application. Although hESC-derived cardiomyocytes (hESC-CMs) hold considerable promise for treatment of cardiac disease, there is a general lack of understanding of how hESC-CMs differentiate in vitro and function in vivo. This application proposes to provide basic information, in murine models, designed to improve the yield of hESC-CMs (using cardiac tissue-specific promoters), to minimize the tumorigenicity and immunogenicity of hESC-CMs, and to monitor hESC-CM survival, trafficking, and efficacy (ie improvement of myocardial function) in vivo. The work proposed is therefore highly significant for hESC replacement therapy of cardiac disease in particular. Although generally responsive to the CIRM SEED Grant RFA, the PI for this project is very senior and has already published a number of directly relevant peer-reviewed publications (9 papers over the last 2 years related to human and murine ESC transplantation for cardiovascular therapies) on the work proposed and appears to already have grant support for at least certain aspects of this work. The proposal therefore may not fulfill the criteria spirit of a “SEED” grant. Although important, it largely represents the continuation of a mature line of research and thus appears more suitable for “RO1”-type, rather than exploratory, funding. Nonetheless, the work proposed is innovative. The PI has formulated and will test the hypothesis that hESC-CMs transplanted into ischemic myocardium can “sense” the ischemic microenvironment and upregulate cell-cycle and angiogenic pathways. If confirmed, this may lead to hESC-mediated cell replacement therapy for treatment of coronary artery disease. The research plan is conceptually and technically sound. The proposed work will leverage the technology and expertise at Stanford in cardiology, cardiothoracic surgery, and molecular/small-animal imaging. It will utilize transduction of hESCs with a triple-reporter fusion construct (FLuc-eGFP-HSVtk) for in vivo bioluminescence imaging, fluorescence microscopy, and PET, respectively, and will include ex vivo tissue analyses to validate the results of molecular imaging studies. Several important components of the research plan are not clear, however. The proposal states (pg 7) that cardiophysiology studies will be performed by treadmill exercise time, US for myocardial contractility, and N13-NH3 and F18-FDG microPET for perfusion and metabolism, respectively. It appears that these parameters will be compared in animals with and without hESC-CMs implanted in the myocardium following some ischemic insult. If there is no such insult (ie transplantation of hESCs only into normally perfused myocardium), the significance of the foregoing measurements is unclear. If there is an ischemic insult, it should be stated and described explicitly. It is also not clear if LV function (EF) will be evaluated and how it will be done (US? gated microPET?). There are bits and pieces of innovation in the proposal, but it is so diffuse, especially for a seed grant, that the applicant does not really emphasize the most innovative aspects. The most innovative part of the application of broad interest to anyone interested in transplantation is the tracking of cells after injection. The ability to understand how stem cells home is critical to translational therapies, and the techniques to follow cells after transplantation are currently very limited. STRENGTHS: One reviewer notes that the major strength of the proposal is the multidisciplinary team it brings together, and finds the preliminary data compelling. The second reviewer finds the potential and long-term implications of hESC cell replacement therapy for cardiac disease highly significant. This reviewer likes the use of postmortem ex vivo tissue analyses, including histopathology, immunohistochemistry, Western blots, and PCR, to confirm/validate the results of molecular imaging studies. There is compelling preliminary data demonstrating the feasibility of the proposed work. The investigative team is outstanding with truly world-class infrastructure in cardiology, cardiothoracic surgery. and molecular and small-animal imaging. A description of statistical analyses is included in the research plan and potential difficulties are acknowledged and alternative plans described. There is extensive intra-institutional collaboration, with signed letters of collaboration provided. WEAKNESSES: The reviewers find the research plan overly ambitious. Any single one of the six sub-aims could take up the entire effort for the grant. Just the differentiation strategies have taken good developmental biologists decades to elucidate, for example getting a handle on the wnt pathway and all its interactions (let alone all the other pathways proposed for examination). For example, one strategy in which the downsides do not seem to be acknowledged sufficiently is the idea of using suicide genes in transplanted cardiomyocytes that can go awry. Is this really a clinically relevant strategy for this particular application? If transplanted cardiomyocytes integrate functionally into host myocardium, what would the potential consequences be of killing off the transplanted cells? (For example, compare the clinical consequences of knocking off transplanted islets vs. transplanted cardiomyoctyes. It seems the latter could be disastrous in terms of arrhythmia or acute myopathy.) The PI has established productive collaborations with the investigators cited in the proposal. One concern is that the basic responsibilites are outlined for each sub-investigator, but it is unclear what part of the stem cell biology Dr. Weissman will supervise, and no genomics are actually mentioned in the grant and that is the expertise cited for Dr. Quertermous (perhaps the MHC studies?) If the names had been connected to aims or something clearer than just the broad areas, the nature of the collaborations would have been clearer for reviewers. The proposal appears to represent the continuation of a mature line of research and therefore may not fulfill the criteria of a “SEED” grant. To one of the reviewers, this is perhaps the most notable weakness of this proposal. There are some apparent ambiguities in the research plan viz-a-viz assessment of cardiophysiology parameters in mice following myocardial implantation of hESC-CMs. Overlap was noted between this proposal and with the work proposed in another the SEED Grant Proposal submitted by (Dr. Joseph Wu) another PI from the same institution. Dr. Wu’s proposal deals specifically with tumor (ie teratoma) formation post-hESC engraftment. The percent effort of the proposal’s professional personnel - only 5% for Dr. Robbins (the Principal Investigator), 5% for Dr. Connolly, and 3% for Dr. Quertermous - is marginally adequate. DISCUSSION: This is an unfocused, overly ambitious, very broad-based proposal that aims to study derived of cardiomyocytes from hESCs and tumorigenicity in models of transplantation. In terms of innovation, the use of suicide genes in transplanted cardiomyocytes is a novelty. Clinically, however, the addition of suicide genes to the cardiomyocytes, while innovative, is irrelevant. Although there is some ambiguity in the research plan, the reviewers were impressed by the stellar team assembled by the applicant and the world class institution that is known for molecular imaging. This proposal is the continuation of a long line of good work, but may not be in the spirit of a SEED grant. The personnel chart was a concern - the listed personnel are not tied to specific aims and it is not clear who is doing what. No collaborator has more than 5% effort listed, which is marginally adequate at best.